Hematopoietic stem cells (HSCs) are the functional units of bone marrow transplantation, which is used in the treatment of variety of blood cell diseases including leukemia and autoimmune disorders. Clinical use of HSCs is limited by the fact that they are rare cells, occurring at a frequency of only <1/20,000 bone marrow cells. Efforts to expand HSCs prior to transplant by ex vivo culturing have proven challenging and thus far such efforts have not translated to the clinic. Thus there remains a clinical need to find alternative strategies for either expanding the numbers of HSCs, or generating HSCs de novo. Numerous studies have shown that it is possible to experimentally reprogram the cellular identity of one cell type to another by enforced expression of transcription factors involved in the specification of the target cell type. The long- term objective of this project is to use this experimental paradigm and express transcription factors involved in specification of HSC fate and function in committed blood cells to reprogram them back to an induced stem cell fate. In order to achieve this we must first identify the factors involved in specifying the fate and function of HSCs. This will be achieved using a large-scale expression profiling strategy designed to allow us to identify transcription factors uniquely expressed in HSCs compared to the other cells of the blood system. Our preliminary studies have identified 28 such genes. Once identified we will then clone these factors into an inducible lentiviral delivery system to allow us to combinatorially express these genes in committed blood cells. We will then test whether or not committed blood cells receiving such factors are able of function like HSCs using a wide variety of stem cell assays. Once reprogramming has been achieved, it will then be important to determine the minimal combination of factors capable of mediating reprogramming. We will further examine the fidelity of reprogramming by functional, molecular and epigenetic analyses. Successful identification of factors capable of reprogramming committed blood cells to an induced HSC fate has the potential to advance our basic understanding of hematopoietic stem cell biology, and contribute to their clinical utility in a number of ways. For example, identification of factors capable of reprogramming committed blood cells to HSCs could provide important insights the molecular mechanisms involved in specifying the fundamental stem cell properties of self-renewal and multi-potency. In terms of clinical application, if successful, our study opens the possibility of eventually generating patient- specific induced HSCs for use in bone marrow transplantation, or for the study of blood disorders. PUBLIC HEALTH RELEVANCE: Although cellular reprogramming strategies have been used to experimentally generate a number of cell types, the generation of hematopoietic stem cells (HSCs) by reprogramming has not yet been described. The goal of our proposal is to identify transcriptional factors with enriched expression in HSCs, and then express these factors in committed blood cells combinatorially to identify factors capable of imparting stem cell properties onto these otherwise committed cells. If successful, our proposal has the potential to provide important mechanistic insight into the central stem cell properties of self-renewal and multi-potency. It would also provide a novel experimental paradigm for generating and studying HSCs.